Recombinant protein expression

ABSTRACT

There are provided methods for the expression of a recombinant protein of interest, said methods comprising, in additional to various additional steps: a) culturing a host cell which expresses: i) one or more genes encoding the recombinant protein(s) of interest; ii) at least two genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GRoES, Dnak, DnaJ, GRpe, ClpB and their homologs (for example, Hsp104, Ydj1 and Ssa1 in yeast); under conditions suitable for protein expression; and separating said recombinant protein of interest from the host cell culture. Also provided are methods for increasing the degree of refolding of a recombinant protein of interest by ading a composition containing a chaperone protein to a preparation of the recombinant protein of interest in vitro.

The invention relates to methods for increasing the yield of foldedrecombinant protein in host cells.

All publications, patents and patent applications cited herein areincorporated in full by reference.

The overproduction of recombinant proteins in cellular systemsfrequently results in misfolding of these proteins. The fates of themisfolded recombinant proteins differ. They may refold to the nativestate or be degraded by the proteolytic machinery of the cell or bedeposited into biologically inactive large aggregates known as‘inclusion bodies’.

The folding of proteins and the refolding of misfolded soluble andaggregated proteins is known to be mediated by a network ofevolutionarily conserved protein molecules called chaperones (Hartl, F.U., Nature, 381, 571-580, (1996); Horwich, A. L., Brooks Low K., Fenton,W. A., Hirshfield, I. N. & Furtak, K., Cell 74, 909-917 (1993); Ellis,R. J. & Hemmingsen, S. M., TiBS, 14, 339-342, (1989); Bukau, B.,Hesterkamp, T. & Luirink, J., Trends Cell Biol., 6, 480-486, (1996);Bukau, B., Deuerling, E., Pfund, C. & Craig, E. A., Cell, 101, 119-122,(2000)). Major chaperones include members of evolutionarily conservedprotein families, including the Hsp60 family (which includes thebacterial chaperone GroEL), the Hsp70 family (which includes thebacterial chaperone DnaK), the Hsp100 family (which includes thebacterial chaperone ClpB), the Hsp90 family (which includes thebacterial chaperone HtpG), the bacterial Trigger factor family, and thesmall HSPs (which includes the bacterial proteins IbpA and IbpB).

Bacterial systems like the gram-negative bacterium Escherichia coli area popular choice for the production of recombinant proteins. In E. coli,it is known that the DnaK and GroEL/ES chaperone systems assist the denovo folding of proteins (Hartl, F. U., Nature, 381, 571-580, (1996);Ewalt, K. L., Hendrick, J. P., Houry, W. A. & Hartl, F. U. Cell 90,491-500 (1997); Bukau, B., Deuerling, E., Pfund, C. & Craig, E. A.,Cell, 101, 119-122, (2000); Teter, S. A. et al., Cell, 97, 755-765,(1999); Bukau, B. & Horwich, A. L, Cell, 92, 351-366, (1998); Deuerling,E., Schulze-Specking, A., Tomoyasu, T., Mogk, A. & Bukau, B. Nature 400,693-696 (1999)).

Furthermore, DnaK and its co-chaperones DnaJ and GrpE are presentlyconsidered to form the most efficient chaperone system for preventingthe aggregation of misfolded proteins (Mogk, A. et al., EMBO J., 18,6934-6949, (1999); Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P. &Bukau, B., Mol, Microbiol., 40, 397-413, (2001); Gragerov, A. et al.,Proc. Natl. Acad. Sci. U.S.A. 89, 10341-10344 (1992)). Increased levelsof GroEL and its co-chaperone GroES have been shown to prevent the heatinduced aggregation of proteins in cells deficient of other majorchaperones (Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P. & Bukau,B., Mol, Microbiol., 40, 397-413, (2001); Gragerov, A. et al., Proc.Natl. Acad Sci U.S.A. 89, 10341-10344 (1992)).

Moreover, the disaggregation of protein aggregates in E. coli usingchaperones has been proven for many different cellular proteins in vivo(Mogk, A. et al., EMBO J., 18, 6934-6949, (1999)), as well as in vitrousing thermolabile malate dehydrogenase (MDH) as a reporter enzyme(Goloubinoff, P., Mogk, A., Peres Ben Zvi, A., Tomoyasu, T. & Bukau, B.,Proc. Natl. Acad Sci., USA 96, 13732-13737, (1999)). Proteindisaggregation is achieved by a bi-chaperone system, consisting of ClpBand the DnaK system. Large aggregates of MDH could be resolubilised invitro and MDH was refolded afterwards into its native structure.Importantly, only the combination of both chaperones is active inresolubilisation and refolding of aggregated proteins. A recentpublication showed that the resolubilisation of recombinant proteinsfrom aggregates in vivo is possible. In these experiments, proteinaggregates were generated by temperature upshift, and the solubilisationand refolding of these proteins was measured in the presence of proteinsynthesis inhibitors to ensure that only the pre-existing aggregatedproteins were monitored. Molecular chaperones were able to resolve theaggregates under these conditions.

Previous studies also indicate that the solubility and yield ofrecombinant proteins could be enhanced by the overproduction ofchaperones. Co-overproduction of GroEL/GroES enhanced the solubility ofseveral recombinant proteins synthesised in E. coli (human ORP150, humanlysozyme, p50^(csk) protein tyrosine kinase, phosphomannose isomerase,artificial fusion protein PreS2-S′-β-galactosidase) (Amrein, K. E. etal., Proc. Natl. Acad Sci., USA 92, 1048-1052 (1995); Nishihara, K.,Kanemori, M., Yanagi, H. & Yura, T., Appl. Environ. Microbiol., 66,884-889 (2000); Thomas, J. G. & Baneyx, F., Mol. Microbiol. 21,1185-1196 (1996); Proudfoot, A. E., Goffin, L., Payton, M. A., Wells, T.N. & Bernard, A. R. Biochem J 318, 437442. (1996); Dale, G. E.,Sch{fraction (o)}nfeld, H. J., Langen, H. & Stieger, M., ProteinEngineering, 7, 925-931 (1994)). The overproduction of the DnaK systemtogether with recombinant target proteins elevates the solubility ofendostatin, human ORP150, transglutaminase and the fusion proteinPreS2-S′-β-galactosidase (Nishihara, K., Kanemori, M., Yanagi, H. &Yura, T., Appl. Environ. Microbiol., 66, 884-889, (2000); Thomas, J. G.& Baneyx, F., J Biol Chem 271, 11141-11147 (1996); Yokoyama, K.,Kikuchi, Y. & Yasueda, H., Biosci. Biotechnol. Biochem. 62, 1205-1210(1998)).

So far, no systematic approach has been made, to analyse whether thecombination of all three chaperones systems (DnaK, DnaJ GrpE; GroES,GroEL and ClpB) expressed together with target genes in E. coil cellsenhances solubility of recombinant proteins. Furthermore, none of theabove-described studies allows the widespread optimisation of expressionsystems that is required to improve yields of soluble proteins on ageneral level. For example, each of the prior investigations focused ononly one or a very small number of target proteins. These investigationsalso focused on the use of only one or two combined chaperone systems.In addition, none of these investigations addressed the issue of theimportance of the ratio of the chaperones to one another and to therecombinant target protein. The previous studies therefore did notprovide any understanding of the relationship between differentchaperone proteins with respect to the folding/refolding of recombinanttarget proteins.

Accordingly, there remains a great need in the art for a general methodto improve the yield of soluble recombinant protein in a givenexpression system. Such a method would allow the optimisation ofexpression systems to give maximal yields of soluble target proteins,and be of obvious industrial and commercial benefit.

The present invention is based upon the systematic engineering of cellsfor the controlled co-overexpression of different combinations ofchaperone genes and target genes. In addition, it was investigatedwhether in vivo disaggregation and refolding of recombinant proteinsfrom aggregates/inclusion bodies could be stimulated by enhanced levelsof chaperones when the production of the target protein is stopped. As aresult, the invention provides novel methods of optimising a givenexpression system in order to achieve higher yields of the desiredsoluble recombinant protein.

According a first aspect of the present invention, there is provided amethod for the expression of a recombinant protein of interest, saidmethod comprising:

-   -   a) culturing a host cell which expresses:        -   i) one or more genes encoding one or more recombinant            protein(s) of interest;        -   ii) at least two genes encoding proteins selected from the            group consisting of the chaperone proteins GroEL, GroES,            DnaK, DnaJ, GrpE, ClpB and their homologs (for example,            Hsp104, Ydj1 and Ssa1 in yeast); under conditions suitable            for protein expression; and    -   b) separating said recombinant protein of interest from the host        cell culture.

Through the recombinant engineering of host cells in this manner, theinvention provides novel methods for producing a recombinant protein ofinterest, which have been found to lead to significant improvements inthe levels of protein produced in the system. The mechanism is thoughtto be through increasing the folding rates of particular proteins usingthe co-expression of particular chaperones in controlled amounts. Usingthis system, very high yields of the desired soluble recombinantproteins of interest can be obtained.

Any recombinant protein of interest may be produced using the system ofthe invention. Preferred examples of proteins of interest will beapparent to the skilled reader. Particularly preferred recombinantproteins are those for which it is desirable to produce a large amount,and those of commercial interest.

Furthermore, the invention is readily applicable to a wide range ofknown expression systems by alterations in the cell culture techniquesemployed. For example, anaerobic fermenter-based cell culture would beappropriate for the culture of obligate anaerobes, whereas standardaerobic cell culture techniques would be appropriate for obligateaerobes. The nutrient composition of the culture medium may also bevaried in accordance with the chosen expression system. The mostsuitable method of cell culture for a given expression system will bereadily apparent to the skilled man.

Preferably, the genes selected in step a) ii) include DnaK, DnaJ andGrpE or homologs thereof, and may additionally include ClpB or a homologthereof

In another preferred aspect of the invention, the genes selected in stepa) ii) include GroES and GroEL or homologs thereof.

More preferably, the genes selected in step a) ii) include the DnaK,DnaJ, GrpE, ClpB, GroES and GroEL genes or homologs thereof.

The above combinations of chaperone proteins have been found to beparticularly suitable for use in the methods according to the invention.

According to a further embodiment of the first aspect of the presentinvention, there is provided a method for the expression of arecombinant protein of interest, said method comprising:

-   -   a) culturing under conditions suitable for protein expression a        host cell which expresses:        -   i) one or more genes encoding one or more recombinant            protein(s) of interest;        -   ii) one or more genes encoding proteins selected from the            group consisting of the chaperone proteins GroEL, GroES,            DnaK, DnaJ, GrpE, ClpB and their homologs (for example,            Hsp104, Ydj1 and Ssa1 in yeast);        -   iii) one or more genes encoding proteins selected from the            group consisting of the small heatshock proteins of the IbpA            family and/or the IbpB family and/or their homologs; and    -   b) separating said recombinant protein of interest from the host        cell culture.

The inclusion of a small heatshock protein of the IbpA family and/or theIbpB family with one or more of the chaperone proteins GroEL, GroES,DnaK, DnaJ, GrpE, ClpB in a host cell with a gene encoding a protein ofinterest has been shown to bestow significant beneficial effects on thelevel of expression of the recombinant protein.

For the purposes of this patent specification, two genes or proteins aresaid to be ‘homologs’ if one of the molecules has a high enough degreeof sequence identity or similarity to the sequence of the other moleculeto infer that the molecules have an equivalent function. ‘Identity’indicates that at any particular position in the aligned sequences, theamino acid or nucleic acid residue is identical between the sequences.‘Similarity’ indicates that, at any particular position in the alignedsequences, the amino acid residue or nucleic acid residue is of asimilar type between the sequences. Degrees of identity and similaritycan be readily calculated (Computational Molecular Biology, Lesk A. M.,ed., Oxford University Press, New York, 1988; Biocomputing, Informaticsand Genome Projects, Smith, D. W., ed., academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, New Jersey, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991).

The chaperone proteins for use in the invention therefore includenatural biological variants (for example, allelic variants orgeographical variations within the species from which the genes arederived) and mutants (such as mutants containing nucleic acid residuesubstitutions, insertions or deletions) of the genes. For the purposesof this application, greater than 40% identity between two polypeptidesis considered to be an indication of functional equivalence. Preferredpolypeptides have degrees of identity of greater than 70%, 80%, 90%,95%, 98% or 99%, respectively. It is expected that any protein thatfinctions effectively as a chaperone, or as part of a chaperone system,within the host cells of the expression system will be of value in thedescribed methods.

Preferably, the levels of the respective chaperone proteins arecontrolled in conjunction with the methods described above. Preferably,the levels of chaperone proteins are controlled by expressing the genesencoding the respective chaperone proteins from different promoters.Preferably, a selection or all of the promoters used are inducible.Different promoters may have different strengths and may respond to thesame induction agent with different kinetics or be responsive to adifferent induction agent, allowing independent control of theexpression level of each chaperone protein. Suitable promoters will beapparent to those of skill in the art and examples are given in standardtextbooks, including Sambrook et al., 2001 (Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.); Ausubel et al., 1987-1995 (Current Protocols in MolecularBiology, Greene Publications and Wiley Interscience, New York, N.Y.).Examples of suitable promoters include IPTG-regulated promoters, such asthe PA11 and lac-O1 promoters (see Tomoyasu, 2001).

Alternatively, or in addition, the respective chaperone proteins areexpressed using expression systems of different strength. Examples ofdifferent expression systems will be clear to those of skill in the art;discussion of such systems may be found in standard textbooks, includingSambrook et al., 2001 (supra) and Ausubel et al., (supra). For example,the plasmid vector of the expression system may be a high copy number orlow copy number plasmid. For instance, examples of E. coli compatiblelow copy number plasmids include pSC101 and p15A ori.

Preferably, the chaperone proteins are over-expressed relative to theexpression levels that occur naturally in non-recombinant cells.

Similarly, the invention provides for the levels of the chaperoneproteins relative to the recombinant protein(s) of interest to becontrolled by expressing the genes encoding the respective proteins fromdifferent promoters, for the reasons described above. For example, in asystem that utilises an IPTG-inducible promoter for expression ofchaperone proteins, an arabinose-inducible promoter may be used tocontrol expression of the recombinant protein of interest. In addition,the expression of the chaperones and of the recombinant proteins(s) canbe controlled using different polymerases.

In a second aspect, the invention also provides methods comprising theuse of a block in protein synthesis during the culturing steps a)described above. Preferably, the block in protein synthesis is imposedby addition of an effective amount of a protein synthesis inhibitor tothe culture system, once a desired level of recombinant protein ofinterest has accumulated. More preferably, the chosen protein synthesisinhibitor is chloramphenicol, tetracycline, gentamycin or streptomycin.In order to ensure that protein synthesis is adequately inhibited, aneffective amount of a protein synthesis inhibitor should be added.Details of effective amounts of protein synthesis inhibitor will beapparent to the skilled reader and are noted in standard textbooks. Forexample, for use in prokaryotic host cell systems, 200 μg/mLchloramphenicol is effective to inhibit protein synthesis.

Any other method that inhibits protein synthesis may also be of valuefor use with the methods of the invention. This includes the use ofmutant strains that are conditionally defective in protein synthesis,for example because of the temperature sensitivity of an enzyme involvedin plasmid or host cell DNA replication or in target gene and host genetranscription or in protein translation. The imposition of such a blockin protein synthesis has been found to lead to significant increases inthe level of recombinant protein that is generated in the system of theinvention.

Alternatively, or in addition, the invention also provides for the useof a reduction in gene transcription, by removal of any agents that areeffective to induce recombinant protein expression (such as IPTG for Lacrepressor controlled genes), once a desired level of recombinant proteinof interest has accumulated. Alternatively, a reduction of constructtranscription could be achieved via the addition of a transcriptionblocking compound (such as glucose for catabolite repressable genes).

This aspect of the invention thus provides a method for the expressionof a recombinant protein of interest, said method comprising:

-   -   a) culturing a host cell which expresses:        -   i) one or more genes encoding one or more recombinant            protein(s) of interest;        -   ii) one or more genes encoding one or more proteins selected            from the group consisting of the chaperone proteins GroEL,            GroES, DnaK, DnaJ, GrpE, ClpB and their homologs (for            example, Hsp104, Ydj1 and Ssa1 in yeast); under conditions            suitable for protein expression;    -   b) imposing a block in protein synthesis, for example by        addition of an effective amount of a protein synthesis inhibitor        to the culture system, once a desired level of recombinant        protein of interest has accumulated; and    -   c) separating said recombinant protein of interest from the host        cell culture.

Also provided is a method for the expression of a recombinant protein ofinterest, said method comprising:

-   -   a) culturing a host cell which expresses:        -   i) one or more genes encoding one or more recombinant            protein(s) of interest;        -   ii) one or more genes encoding one or more proteins selected            from the group consisting of the chaperone proteins GroEL,            GroES, DnaK, DnaJ, GrpE, ClpB and their homologs (for            example, Hsp104, Ydj1 and Ssa1 in yeast); under conditions            suitable for protein expression;    -   b) imposing a reduction in gene transcription, for example by        removal of any agents that are effective to induce recombinant        protein expression (such as IPTG for Lac repressor controlled        genes), or via the addition of a transcription blocking compound        (such as glucose for catabolite repressable genes), once a        desired level of recombinant protein of interest has        accumulated; and    -   c) separating said recombinant protein of interest from the host        cell culture.

One or more genes encoding proteins selected from the group consistingof the small heatshock proteins of the IbpA family and/or the IbpBfamily and/or their homologs may also be included in the host cell. Theinclusion of such proteins in conjunction with the imposition of areduction in gene transcription or the imposition of a block in protiensynthesis.

Preferably, a combination of chaperone proteins is expressed asdescribed above.

Preferably, the chaperone proteins are expressed under a differentpromoter to that used to control expression of the recombinant proteinof interest.

Preferably, the chosen protein synthesis inhibitor is chloramphenicol,tetracycline, gentamycin or streptomycin.

Preferably, in the methods of the above-described aspects of theinvention the cultured host cell is a prokaryotic cell, such as an E.coil cell, a Lactococcus cell, a Lactobacillus cell or a Bacillussubtilis cell, or a eukaryotic cell such as a yeast cell, for example aPichia or Saccharomyces yeast cell, or an insect cell, for example afterbaculoviral infection.

Preferably, an optimised yield of recombinant protein of interest ismanifested by increasing the level of de novo protein folding.

An optimised yield of said recombinant protein of interest may also bemanifested by increasing the level of in vivo refolding of aggregated,or misfolded soluble, recombinant protein.

An optimised yield of said recombinant protein of interest may also bemanifested by increasing the level of in vitro refolding of aggregated,or misfolded soluble, recombinant protein.

An optimised yield of said recombinant protein may also be manifested byincreasing the level of de novo protein folding in combination withincreasing the increased level of in vivo refolding and/or in vitroprotein refolding.

Preferably, said increased level of folding or refolding results inincreased solubility of the recombinant protein of interest.

Preferably, said increased level of folding or refolding results inincreased activity of the recombinant protein of interest.

According to a third aspect of the present invention there is alsoprovided a method for increasing the degree of refolding of arecombinant protein of interest, said method comprising adding acomposition containing a chaperone protein to a preparation of therecombinant protein of interest in vitro. This has been found toincrease significantly the degree of refolding of protein inpreparations containing wholly or partially unfolded protein. Thepreparation of the recombinant protein of interest may be anypreparation that contains protein that is partially or wholly unfoldedor misfolded. Preferably, the preparation is a cell extract preparation,such as a lysate of a prokaryotic cell.

Preferably, a combination of chaperone proteins as described above isadded to the preparation of the recombinant protein of interest. Forexample, such chaperone proteins may include one or more genes encodingone or more proteins selected from the group consisting of the chaperoneproteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs (forexample, Hsp104, Ydj1 and Ssa1 in yeast), and optionally one or moregenes encoding proteins selected from the group consisting of the smallheatshock proteins of the IbpA family and/or the IbpB family and/ortheir homologs.

The preparation of the recombinant protein of interest may be apreparation of soluble recombinant protein that has been precipitated invivo, or may be a preparation of in vitro precipitated recombinantprotein (for example, a host cell extract containing the recombinantprotein aggregate).

Preferably, said composition containing the chaperone protein(s) isadded after removal of any agents that are effective to induce solublerecombinant protein expression (such as IPTG for Lac repressorcontrolled genes) or after addition of a transcription blocking compound(such as glucose for catabolite repressable genes).

Preferably, the third aspect of the invention is used in conjunctionwith imposing a block in protein synthesis, for example by addition ofan effective amount of a protein synthesis inhibitor to the culturesystem. As described above, chloramphenicol, tetracycline, gentamycinand streptomycin are examples of suitable protein synthesis inhibitors.

Preferably, when practising the above-described methods, the time courseof refolding and the temperature at which refolding occurs iscontrolled. The time course of refolding and temperature at which itoccurs are known to have a significant effect on the yield of solublerecombinant protein, and are thus an important aspect of a givenexpression system to be optimised for the maximal yield of solublerecombinant protein.

Preferably, when practising the above-described methods, a compositioncontaining a protein selected from the group consisting of the smallheatshock proteins of the IbpA family and/or the IbpB family and/ortheir homologs is used in conjunction with the chaperone proteins GroEL,GroES, DnaK, DnaJ, GrpE, and/or ClpB and/or their homologs.

A further aspect of the present invention relates to methods for theprophylaxis, therapy or treatment of diseases in which aggregatedproteins are implicated, comprising the administration of the describedcombinations of chaperone proteins and/or small heatshock proteins insufficient amounts. Such diseases include, but are not limited todiseases in which amyloid deposits are implicated, such as late andearly onset Alzheimer's disease, SAA amyloidosis, hereditary Icelandicsyndrome, multiple myeloma, and spongiform encephalopathies.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example. It will be appreciated thatmodification of detail may be made without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows chaperone co-overproduction systems tested in E. coli.Genes encoding three different chaperone-systems (GroEL/ES; DnaK, DnaJ,GrpE; and ClpB) were cloned in a pair of low copy number vectors, whichare compatible with E. coli (SC101 and p15A ori), carry the lacI^(Q)gene and different resistance markers for selection. Chaperone genes areset under the control of IPTG-regulated promoters (PA11/lacO1) forcontrolled expression. Each combination of vector pairs (1 to 5) differsin its combination and level of chaperone expression. In these strainssubsequently a third plasmid encoding a substrate protein wasintroduced.

FIG. 1B shows chaperone expression patterns. The chaperone combinations2 to 5 are shown. The left hand lane of each pair is loaded with asample for which expression of the recombinant proteins had not beeninduced. The right hand column for each chaperone combination shows anIPTG-induced sample.

FIG. 2. Chaperone and target protein co-expression under IPTG control.The target proteins Tep4, Btke and Lzip were purified by metal affinitychromatography after transformation in BL21(DE3) cells used as a control(K) and in the same strain but co-expressing the 5 different chaperonecombinations reported in FIG. 1.

FIG. 3. In vivo induced refolding. FIG. 3A shows the Btke expressionlevel after chaperone-induced re-folding in BL21(DE3) cells used as acontrol (K) and in the same strain but co-expressing the 5 differentchaperone combinations reported in FIG. 1. Cells were grown at 30° C.,induced with 0.1 mM IPTG, grown overnight, and then either grown 2 morehours (first lane of each combination) or pelletted, re-suspended infresh medium plus 200 μg/mL chloramphenicol and cultured 2 more hours(second lane). FIG. 3B shows optimisation of the re-folding conditionsusing the chaperone combination 4 shown in FIG. 3B. After overnightculture at 20° C. the cells were pelletted, resuspended in fresh mediumand cultured 1 h, 2 h, 3 h, and 4 h at 20° C. (1 to 4), or 1 h and 2 hat 37° C. (5 and 6) in the presence of 200 μg/mL chloramphenicol. Foreach combination the first lane was loaded with the uninduced sample andthe second with the treated one. FIG. 3C shows Btke expressed in control(C1) and chaperone combination 4 (C2) cells. Lanes were loaded withuninduced samples (K), induced and cultured at 20° C. overnight plus twohours at the same temperature, pelleted after overnight growth,resuspended in fresh medium plus 200 μg/mL chloramphenicol and cultured2 more hours, as in 2 but in the presence of 1 mM IPTG instead ofchloramphenicol, resuspended in fresh medium for 1 h, 2 h, and 4 h. Thenumbers shown below the gel image indicate the increase factor obtainedcomparing the intensity of the bands to the reference (induced cellswithout chaperone co-expression). FIG. 3D shows the effect of growthconditions on re-folding efficiency of Btke. Cells were grown overnightat 20° C. (D1) and at 42° C. before inducing the re-folding at 20° C.(D2). Lanes were loaded with un-induced samples (K), induced andcultured overnight plus two hours (1), resuspended in fresh medium plus2 h culture (2), in fresh medium plus 200 μg/mL chloramphenicol andcultured 2 more hours (3). FIG. 3E shows the re-folding efficiency ofTep4 expressed in control (E1) and chaperone combination 4 (E2) cells.Lanes were loaded with uninduced samples (K), induced and culturedovernight plus two hours (1), resuspended in fresh medium plus 2 hculture (3), in fresh medium plus 200 μg/mL chloramphenicol and cultured2 more hours (4).

FIG. 4. In vitro re-folding. FIG. 4A shows Btke expressed either incontrol cells (c) or in cells co-expressing chaperone combination 3 or4. 3 h after IPTG induction, cells were harvested and lysate prepared asdescribed above. Samples containing 100 μg lysate were supplemented with10 mM ATP and 3 mM PEP and 20 ng/ml PK. After indicated timepoints,soluble Btke protein was isolated and analysed by SDS-PAGE and Coomassiestaining. FIG. 4B shows the results produced when pellets with insolubleBtke were isolated from control cells. Pellets were suspended in bufferand where indicated chaperones were added. After 5 min, 2, 4, and 20 hsoluble Btke protein was isolated as described above and analysed bySDS-PAGE and silver staining.

FIG. 5 shows the results of experiments to test the effects of variouscombinations of different sHSPs and HSPs on the refolding of soluble MDHcomplexes in vitro.

FIG. 6 shows the results of experiments to test the effects of differentHSP combinations on the refolding of soluble α-glucosidase/sHSP 16.6 andcitrate synthase/sHSP 16.6 complexes in vitro.

FIG. 7 shows the results of experiments to test the effects of differentHSP combinations on the refolding of aggregated luciferase and solubleluciferase/sHSP 16.6 complexes in vitro

FIG. 8 shows the results of KJE/ClpB-mediated refolding of MDH. Thedifferent 16.6 concentrations present during MDH denaturation are shownas the indicated 16.6/MDH ratio. Refolding curves for KJE-mediatedrefolding of MDH are indicated. Refolding curves for refolding of MDHcarried out in presence of ClpB/DnaK are differently coloured. Theprecise 16.6/MDH ratios during MDH denaturation are indicated to theright of the graph and are as follows: green (16.6/MDH ratio=0); lightblue (16.6/MDH ratio=0.25); brown (16.6/MDH ratio=0.5); dark blue(16.6/MDH ratio=1); yellow (16.6/MDH ratio=2); pink (16.6/MDH ratio4).

FIG. 9 shows the results of experiments to determine the effect onprotein refolding of varying the concentration of ClpB.

FIG. 10 shows the results of experiments to determine the effects ofmutations to the ibpAB genes and DnaK genes of E. coli.

FIG. 11 shows a comparison between the effects of mutations to the ibpABand clpB genes in E coli on the thermotolerance of those strains.

FIG. 12 shows the results of experiments to determine whether IbpA/Bprotein function increases in importance in the presence of reducedlevels of DnaK and at elevated temperatures.

FIG. 13 shows the results of experiments to determine the levels ofprotein aggregation associated with heat shock in ΔibpAB ΔclpB doubleknockout E. coli cells.

FIG. 14 shows the effect of IpbAB co-expression on the level of solubletarget proteins produced in E. coli cells.

FIG. 15: Effect of plasmid interactions on the level of the recombinantprotein expression. A) Recombinant chaperone (K-DnaK, ELS-GroELS, ClpB)accumulation in bacteria homogenates. B) Accumulation of co-expressedrecombinant chaperones and target protein GTR1 in the homogenatesrecovered from control (C) and induced (I) bacteria. C) Effect ofchaperone co-transformation on the not induced (C) and IPTG-induced (I)expression of the target protein Btk cloned in pET24d. D) Effect of theco-transformation with an empty pDM1 vector on the not inducedexpression of Btk cloned in pET24d.

FIG. 16: Co-expression of the coil-coiled region of Xklp3A/B. The chainsA and B were cloned in a polycistronic vector and expressed either inBL21 (DE3) together with the recombinant chaperone combinationK+J+E+ClpB+GroELS (+chap) or in BL21 (DE3) pLysS in the presence of 1%glucose (−chap).

FIG. 17: Effect of unsynchronised recombinant chaperone expression onthe level of soluble target recombinant protein. The independentinduction of the chaperones and target proteins has been obtained usingarabinose-regulated vectors for the target proteins and IPTG-induciblevectors for the chaperones. In the figures are reported the bandscorresponding to the soluble target protein purified by affinitychromatography from 0.5 20 mL of bacterial culture. A) Amount of solubleGTR1 and coiled-coil Xklp3A recovered from wild type cells and bacteriaco-transformed with different chaperone combinations. Expression wasinduced by 0.2 mM IPTG and 1.5 mg/mL arabinose were added 20 min later.The samples were collected 3 hours after the IPTG induction. B) Amountof soluble GTR1 recovered from bacteria co-transformed withK+J+E+ClpB+GroELS and using different combinations of time andexpression-inducer concentrations. The samples were collected 3 hoursafter the addition of the first inducer and the bands corresponding toGroEL are recovered from SDS-gels loaded with the soluble fraction aftercell lysis. C) Amount of soluble coiled-coil Xklp3B recovered frombacteria co-transformed with K+J+E+ClpB+GroEL after overnight culture(ON) at 20° C. The replacement of the ON medium with fresh medium (Fr.Md.), 0.2 mM chloramphenicol (Chlor.) and the temperature shift to 30°C. were used to stimulate the in vivo re-folding of the aggregatedtarget protein.

EXAMPLES

Examples 1-5 below illustrate the materials and methods used toinvestigate the effect of co-expressing different chaperone combinationson the yield of a large variety of different recombinant proteins.

Example 1 Construction of Chaperone Vectors

Plasmids carrying chaperone genes under the control of theIPTG-sensitive promoter PA1/lacO-1 were constructed as described(Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P., Buckau, B., Mol.Microbiol., 40, 397-413, (2001)). Target protein vectors were deliveredto the Protein Expression Unit from different research groups working atthe European Molecular Biology Laboratory.

Example 2 Transformation Procedure

Competent BL21 (DE3) and Top10 cells were transformed with the followingcouples of plasmids for selective expression of chaperone combinations(FIG. 1A). The clones for DnaK, DnaJ, and GrpE were carried by pBB530and pBB535; the co-expression of DnaK, DnaJ, GrpE, and ClpB wasregulated by pBB535 and pBB540; GroEL/ES system was expressed by pBB528and pBB541; a large amount of the complete system DnaK, DnaJ, GrpE,ClpB, and GroEL/ES was ensured by pBB540 and pBB542; finally, a lowerexpression level of the same chaperone combination was obtained usingpBB540 and pBB550. A complete array of single chaperone plasmidtransformed cells was also prepared as a control. Transformed cells werechecked for chaperone expression and successively made competent. Theprotease deficient strain BB7333 (MC4100 ΔclpX, ΔclpP, Δlon) was usedfor transforming the Btkp protein. These strains were also madecompetent and used for a further transformation with the targetproteins.

Example 3 Cell Cultures

Single colonies from the transformed cells were used to inject 3 mL ofLB medium. Liquid cultures were performed initially at 37° C., thentransferred to 30° C. and finally transferred to 20° C. Using differenttimes of incubation at the higher temperatures it was possible to reachthe OD₆₀₀ of 0.8 at the same time for all the different cell strainscultured together for comparative expression assays. Protein expressionwas performed overnight by inducing gene transcription using 0.1 mMIPTG. 1.5 mL of the overnight culture of both IPTG-induced (hereaftertermed ‘induced) and control bacteria was directly centrifuged in anEppendorf tube and the pellet frozen and stored at −20° C.Alternatively, the pellet was re-suspended in 3 mL of fresh medium anddivided into two aliquots of 1.5 mL, with or without the addition of 200μg/mL chloramphenicol. After 2 h culture at 20° C. the cells wereharvested as described before. Inclusion body overproduction wasobtained by culturing the bacteria at 42° C. overnight after induction.Large scale cultures were grown in 2 L flasks using 5 mL of overnight LBpre-culture to inoculate 500 mL of Terrific Broth.

Example 4 Protein Purification and Evaluation

Frozen bacterial pellets were re-suspended in 350 μL of 20 mM Tris HCl,pH 8.0, 2 mM PMSF, 0.05% Triton X-100, 1 μg/mL DNAase and 1 mg/mLlysozyme and incubated on ice for 30 min, with periodic stiring. Thesuspension was sonicated in water for 5 minutes, an aliquot (ofhomogenate) was stored and the rest was pelleted in a minifuge. Analiquot of the supernatant was preserved and the rest was added to 15 μLof pre-washed magnetic beads (Qiagen) and incubated further 30 min underagitation before being removed. Beads were washed 30 min with 20 mMK-phosphate buffer, pH 7.8, 300 mM NaCl, 20 mM imidazole, 8% glycerol,0.2% Triton X-100 and later with PBS buffer plus 0.05% Triton X-100.Finally they were boiled in 12 μL SDS sample buffer and the samplesloaded for SDS PAGE analysis, using a Pharmacia minigel system. Proteinswere detected after coloration with Simply Blue Safestain (Invitrogen)following the manufacturer's instructions and the gels were recordedusing a Umax Astra 4000U scanner. Bands corresponding to the proteinswere analysed using the public NIH Image 1.62f software. Alternatively,protein was eluted from washed beads using 30 μL PBS buffer plus 0.5Mimidazole and its relative concentration measured following itsadsorbance at 280 nm. The proper folding was evaluated by circulardichroism using a J-710 spectropolarimeter (Jasco).

Example 5 In vitro Experiments

Cells were grown in LB and after inducing the synthesis of either Btkeor Btke together with GroEL/ES (combination 3) or together withGroEL/ES, DnaK, DnaJ, GrpE, ClpB (combination 4) for 3 h with 1 mM IPTGat 37° C., lysates were prepared as described above. For refolding ofBtke from inclusion bodies using total lysate, 10 mM ATP, 3 mMphosphoenole pyruvate (PEP) and 20 ng/ml pyruvate kinase (PK) were addedand incubated at 20° C. After 5 min, 2, 4, and 20 h soluble material wasseparated from insoluble fractions by centrifugation (15 min, 4° C.,10.0000 rpm) and the soluble fraction was used to isolate target proteinas described above.

For resolubilsation of isolated Btke aggregates with exogenous chaperoneaddition, 100 μg of total lysate (isolated from cells with overproducedBtke) was centrifuged for 15 min and pellets were resuspended in 20 mMTris/HCl, 100 mM KCl and 20 mM MgCl. Chaperones were added as indicatedand samples incubated at 20° C. for 5 min, 2, 4, 20 h. Soluble materialwas separated from inclusion bodies by centrifugation and isolated asdescribed.

Examples 6 to 9 below illustrate the optimisation of chaperoneco-expression combinations and other experimental variables in order togreatly increase the yield of a large number of diverse recombinantproteins.

Example 6 Investigation of the Effect of Chaperone Combinations on denovo Protein Folding

Five different combinations of plasmids encoding chaperone systems(GroEL/ES; DnaK, DnaJ, GrpE and ClpB) in different combinations andamounts under the control of IPTG regulated promoters were introducedinto BL21 (DE3) cells as illustrated in FIG. 1A. The degree of chaperoneexpression was shown to be very high (FIG. 1B). These cells weresubsequently transformed with plasmids expressing substrate proteins inan IPTG controlled manner (FIG. 1A). Therefore, co-expression ofchaperones and target proteins was obtained by simultaneous induction ofall the promoters with IPTG. Co-expression of chaperones together with50 different target genes was tested. For each target protein, all fivedifferent chaperone combinations were tested and solubility of therecombinant proteins analysed. In summary a higher yield of solublesubstrate protein was achieved in more than 50% of the tested constructs(see Table 1 below).

Table 1 shows a list of the proteins used in the survey for analysingthe effect of chaperone co-expression on soluble target protein yield.The table shows the molecular weight of the constructs, the originalorganisms from which they were cloned, whether they corresponded to fulllength proteins (F1) or to domains, expressed alone or fused to apartner (fus), and their cell localisation (cytoplasm, membrane,nucleus, secreted) in vivo. The yield increase factor (IF) induced bythe best chaperone combination is reported under ‘Chap. IF’ and theyield increase factor obtained using the refolding protocol under‘Refolding IF’. The symbol (/) signifies that the experiment has not yetbeen done and (!) that protein has been obtained using constructs thatgave no soluble protein when expressed in wild type bacteria. TABLE 1Protein MW Organism Features Chap. IF Refolding IF GTR1 40 kD S.cerevisiae Fl/cyt 3 3 BtKp 55 kD H. sapiens domain/cyt 0 28 Xpot1 110 kDH. sapiens Fl/cyt 0 / XklpA1 62 kD X. laevis domain/fus/cyt 0 ! XklpB140 kD X. laevis domain/fus/cyt 0 ! HbpH 9 kD H. sapiens domain/cyt 3.53.5 TEVprotease 30 kD TEV domain 3.5 / Pex5p 50 kD H. sapiens domain/cyt0 / UCP1 33 kD R. norvegicus domain/membr 0 0 Transcr Fact 37 kD H.sapiens Fl/cyt 0 0 BtKe 55 kD H. sapiens domain/cyt 4 42 XklpA2 38 kD X.laevis domain/fus/cyt 0 / XklpB2 35 kD X. laevis domain/fus/cyt 0 /XklpA3 72 kD X. laevis domain/fus/cyt 0 / Rolled 43 kD D. melanogasterFl/cyt 4.5 4.5 Lzip 41 kD H. sapiens Fl/cyt ! / 1Ap 52 kD D.melanogaster Fl/nucl 0 / Chip 64 kD D. melanogaster Fl/nucl 0 / dLMO 37kD D. melanogaster Fl/nucl 0 / Tlc 57 kD R. prowazekii Fl/membr 0 / BtKc64 kD H. sapiens Fl/cyt 3 / PhosphK 29 kD H. sapiens Fl/cyt 3 7 Compl.Tep3 47 kD A. gambiae domain/fus 4 / Compl. Tep4 45 kD A. gambiaedomain/fus 3.5 / XklpA4 72 kD X. laevis domain/fus/cyt 2.5 2.5 XklpB3 73kD X. laevis domain/fus/cyt 2.5 2.5 E8R1 58 kD Vaccinia virusFl/membr/fus 7 / Compl. Tep3 70 kD A. gambiae domain/fus/secr 0 11Compl. Tep4 68 kD A. gambiae domain/fus/secr 3.5 13 MaxF 7.5 kD synteticdomain 3 / XklpA5 35 kD X. laevis domain/fus/cyt 0 19 E8R2 85 kDVaccinia virus Fl/membr/fus 5.5 5.5 Susy 90 kD Z. mays Fl/membrane 3 5Mash 91 kD Z. mays Fl/cyt 0 3 PPAT 22 kD E. coli Fl/cyt 0 3 2Ap 54 kD D.melanogaster Fl/nucl ! 3 F10L 45 kD Vaccinia virus Fl/fus 0 0 B1R 47 kDVaccinia virus Fl/fus 3.5 3.5 1Frenge 43 kD D. melanogaster domain/cyt !! Tep1 7 kD A. gambiae domain/secr 3 6 Tep2 11 kD A. gambiae domain/secr0 0 2Frenge 55 kD D. melanogaster domain/fus 0 2 GFP-fusion 95 kD A.victoria Fl/fus/cyt 0 0 2C18 50 kD H. sapiens Fl/fus 3 8 22j21 72 kD H.sapiens Fl/fus ! ! XklpA + B 15 + 17 kD X. laevis domain/complex 2.5 3.5Msl3 14 kD D. melanogaster domain/cyt 2.5 2.5 Mash + Susy 94 + 90 kD Z.mays Fl/complex 3 3 Endostatin 22 kD M. musculus domain/secr 0 0 Kringle30 kD H. sapiens domain/fus 0 0

As can be seen from the ‘Chap. IF’ ratings, soluble target protein yieldincreased between 2.5 and 7-fold. Effects of co-expressed chaperoneswere not limited to a certain type of substrate protein. The targetproteins tested were representative of several different classes,including complexes, soluble, membrane-bound and secreted proteins,full-length, domains and fusion constructs, with a molecular weightspanning from 7.5 to 110 kD, expressed in the cytoplasm and in theperiplasm (Table 1). Moreover, in some cases, like Lzip (see Table 1 andalso FIG. 2), co-expression of chaperones was the only possibility toobtain any soluble protein. Evaluation of the 23 positive casesindicated that the most efficient chaperone combination was the fourth,which expressed all three chaperone systems in large amounts, followedby the third, fifth, first and the second. Nevertheless, as isdemonstrated in the case of Lzip transcription factor where chaperonecombination 1 worked far better than the others, any one chaperonecombination is not necessarily optimal for all target proteins. Thus,despite the systematic approach it was not possible to infer generalrules about the optimal conditions to succeed. No protein class showedbetter results in combination with particular chaperone combinations andno expression vector ensured significantly better yields. The onlyexception was when target proteins were cloned in high copy numbervectors. In such a case no positive result was observed. The competitionfor the protein synthesis machinery could be considered as a reason,since chaperone expression is inhibited when a target protein wasco-expressed and is completely prevented in cells harbouring expressionvectors with pUC origin (data not shown). The results shown in Table 1clearly demonstrate the very large increases in yield possible via theuse of the disclosed methods.

Example 7 Testing the Effect of Co-overexpression of ChaperoneCombinations and Target Proteins on Re-folding of Aggregated ProteinsUsing Chloramphenicol

In the experiments of Example 5 it was often observed that inclusionbodies accumulated even in the presence of overproduced chaperonesincreasing the amount of soluble proteins. A recent paper (Carrio, M. M.and Villaverde, A. FEBS Lett., 489, 29-33 (2001)) showed that solubleproteins could be recovered in vivo from inclusion bodies when theprotein synthesis was blocked by chloramphenicol addition and the wholecellular folding machinery became available for precipitated proteins.Therefore, we investigated the overexpression of chaperones not only forkeeping recombinant proteins soluble but also for increasing there-folding capability of cells. To investigate this further, weco-overexpressed chaperones and target genes as described before.Subsequently, we stopped protein synthesis by the addition ofchloramphenicol. Cells were transferred to fresh media, incubated at 20°C. and resolubilisation of targets had been analysed at different timepoints. In fact, in the case of Btke the chloramphenicol-induced blockof protein synthesis induced a low increment of the soluble recombinantprotein in control cells but an impressive increase when specificchaperone combinations were co-expressed simultaneously with the targetgene prior to the translational arrest (FIG. 3A). It is worthy to notethat for Btke the optimal chaperone combination differed when thesoluble protein accumulated during standard culture conditions and whenprotein synthesis has been blocked (FIGS. 2 and 3A). The choice of timeand temperature conditions during re-folding was crucial for optimisingthe result (FIG. 3B). Longer incubation times or higher temperaturelowered the amount of recovered soluble protein, probably becausedegradation by proteases takes over re-folding activity. As can beenseen in Table 1 above, this method of combining chaperoneco-overexpression with the blocking of protein synthesis resulted in agreat improvement in the yield of recombinant protein in a large numberof the combinations tested.

Example 8 Testing the Effect of Co-overexpression of ChaperoneCombinations and Target Proteins on Re-folding of Aggregated Proteins byReducing Construct Gene Transcription

The protocol used to block protein synthesis, as described in Example 6above, was evaluated by means of experiment. It was found that theoriginal protocol can be simplified and that it was not strictlynecessary to completely prevent protein synthesis in order to inducere-folding, and in fact the cessation of recombinant protein expressionby removing the induction agent (IPTG) was sufficient. In this case thetarget protein could be re-folded to a level comparable to that obtainedin the presence of chloramphenicol but only in the presence of therecombinant over-expressed chaperones (FIG. 3C). For Btke the optimalre-folding conditions enabled the recovery of 42-fold more protein thanin the standard growth conditions using normal BL21 (DE3) cells and thesimplified protocol (without chloramphenicol) gave an increase factor of26. We also tried to induce the inclusion body formation culturing thebacteria at 42° C. and starting the re-folding from a higher amount ofmaterial but the improvement was negligible (FIG. 3D), probablyindicating that the limiting factor is represented from the foldingmachinery or from the cellular degrading metabolism. These two factorsseem to be somehow connected, as illustrated in the case of Tep4. Incontrast to Btke this protein was expressed in soluble form atsufficient levels also at standard culture conditions and chaperoneco-expression induced a limited yield increase (FIG. 3E). Nevertheless,the suppression of IPTG induction by simple exchange with fresh mediumboosted the accumulation of soluble protein in both the strains but onlythe co-expression of recombinant chaperones could ensure the sameresults when chloramphenicol was added. Generally, we observed that theaddition of fresh medium alone was more effective than the combinationof fresh medium and chloramphenicol in strains with wild type chaperoneexpression. This indicates that the removal of the inducer IPTG, and thesubsequent cessation of transcription of the target gene, is sufficientto allow refolding from inclusion bodies. It was a goal of the inventorsto obtain more information about the relationship between proteinre-folding and degradation by transforming our vectors in the proteasedeficient strain BB7333. However, the inventors were not able to raise asufficient number of bacterial colonies. This finding confirmed thegeneral role of proteases in maintaining cell viability (Tomoyasu, T.,Mogk, A., Langen, H., Goloubinoff, P., Buckau, B., Mol. Microbiol., 40,397-413, (2001)) and suggests that a certain degree of proteindegradation must be maintained. It is therefore clear from the aboveexample that a reduction in recombinant target gene transcription canalso allow the refolding of aggregated proteins to proceed, leading togreatly improved yields of the soluble recombinant protein of interest.

Protein synthesis inhibitors other than chloramphenicol, such astetracycline, gentamycin and streptomycin have been tested with similareffects.

Example 9 The Effect of Co-overexpression of Chaperone Combinations andTarget Proteins on Re-folding of Aggregated Proteins in vitro

Next, we analysed whether co-expressed chaperones are capable ofenhancing the refolding of target proteins from inclusion bodies invitro after cell lysis. For that purpose, we induced simultaneouslysynthesis of Btke together with either chaperone combination 3 or 4.Cells were harvested after induction and total lysates containinginclusion bodies and chaperones were isolated. Subsequently anATP-regenerating system was added to the lysates and the soluble proteinwas purified after 5 min, 2 h, 4 h and 20 h. Lysate containing thechaperone mixture 4, which was the most efficient during the in vivorefolding of Btke, showed already 5 min after the addition of ATP thatapproximately all Btke could be recovered in the soluble fraction. Thecontrol lysate, where only Btke was overexpressed, and the lysate withenhanced levels of GroEL/EL showed no significant recovery of solubleBtke (FIG. 4A). It is therefore clear that co-overexpressed chaperonemixtures stimulate re-solubilisation of inclusion bodies from bacterialcell lysates. Refolding of Btke inclusion bodies was also possible whenchaperones were added exogenously to isolated aggregates (FIG. 4B).However, refolding efficiency was much lower and refolding kinetics muchslower, most probably due to the limited amount of added chaperones.This example clearly shows that co-expression of chaperones can alsoincrease the yield of soluble recombinant protein via an enhancement ofthe refolding of target proteins from aggregates/inclusion bodies invitro.

The above Examples 1-8 have clearly shown the value of the methodsprovided by the present invention for increasing protein yield. There-folding protocol applied to the chaperone transformed cells allowedeven higher yields of soluble protein than the simple co-expression withthe target proteins in 8 on 17 cases and, importantly, also gavepositive results also in the case of 8 constructs insensitive to simpleco-expression. Taking all the results together chaperones had a positiveeffect on soluble protein accumulation in 68% of the cases analysed inour survey. The ratio remains basically the same if all the 50constructs are considered (34 positive) or if only the 37 differentproteins are taken in account (24 positive, 65%). It must be remarkedthat such a positive result has been obtained despite the fact that mostof the constructs used in the experiment correspond to sequencesdifficult to be expressed in a soluble form in bacteria, likemembrane-associated or secreted proteins, regions not corresponding tostructural domains or complexes (underlined in Table 1). The advantageof the in vivo disaggregation is that protein refolding follows nativepatterns and, therefore, recovers its native conformation. The correctfolding of some of the proteins was analysed by purification untilhomogeneity followed by circular dichroism analysis, indicating that theproteins had adopted their native conformation after refolding.Importantly, the enzymatic activities of the kinases B1R and F10L, theTEV protease and luciferase were also recovered after re-folding (datanot shown). Larger scale cultures confirmed the trend observed in testcultures, suggesting that the disclosed methods are suitable forindustrial applications. In summary, the invention provides not only amethod for the production of large amounts of soluble recombinantprotein, but also a method for the production of large amounts ofrecombinant protein that is correctly folded and furthermore retains thenative protein's biological activity.

In the following examples 10 and 11, the effect of small heat shockproteins (sHSPs) on the yield of soluble recombinant proteins both invitro and in vivo was investigated. Published data had previously shownthat members of the chaperone family of small heat shock proteins(sHSPs), such as the E. coli family members IbpA and IbpB (IbpAB), canefficiently prevent the aggregation of unfolded proteins, although theywere not shown to exhibit protein refolding activity. In the presentstudy, refolding of substrates from sHSP/substrate complexes is reportedto be dependent on an Hsp70 chaperone system (such as DnaK with its DnaJand GrpE co-chaperones) in a reaction that can be further stimulated bythe GroEL and GroES (GroELS) chaperones.

Example 10 Investigation of the Effect of Small Heat Shock Proteins onthe Yield of Soluble Recombinant Proteins in vitro

The refolding of several recombinant proteins from soluble complexes wastested:

Materials and Methods:

1 μM MDH was denatured in buffer A (50 mM Tris pH 7.5; 150 mM KCl; 20 mMMgCl₂) for 30 min at 47° C. either in the presence of 6 μM 18.1 (pea),or 6 μM IbpB (E. coli), or 4 μM 16.6 (Synechocystis sp.). MDH refoldingwas initiated at 30° C. by adding an ATP regenerating system (2 mM ATP;3 mM PEP; 20 ng/ml pyruvate kinase) and various chaperone combinationsmade up from KJE (1 μM DnaK; 0.2 μM DnaJ; 0.1 ,μM GrpE), ESL (4 μMGroEL; 4 μM GroES) and ClpB (1.5 μM). The results for these experimentsare shown in FIG. 5.

Similarly, 1 μM α-glucosidase or 1 μM citrate synthase were denatured inthe presence of 4 μM 16.6 (Synechocystis sp.) in buffer A for 45 min at50° C. or 47° C., respectively. Protein refolding was initiated at 30°C. by adding an ATP regenerating system (2 mM ATP; 3 mM PEP; 20 ng/mlpyruvate kinase) and various chaperone combinations made up from KJE,ESL and ClpB. The results for these experiments are shown in FIG. 6.

Similarly, 100 nM firefly luciferase was denatured in the absence orpresence of 0,4 μM 16.6 (Synechocystis sp.) in buffer A for 15 min at43° C. Luciferase refolding was initiated at 30° C. by adding an ATPregenerating system (2 mM ATP, 3 mM PEP; 20 ng/ml pyruvate kinase) andvarious chaperone combinations made up from KJE (0.5 μM DnaK; 0.1 μMDnaJ; 0.05 μM GrpE) and ClpB (0.5 μM). The results for these experimentsare shown in FIG. 7.

To investigate the effect of the stoichiometry of the sHSPs on therefolding of sHSP/substrate complexes 1 μM MDH was denatured in buffer A(50 mM Tris pH 7,5; 150 mM KCl; 20 mM MgCl₂) for 30 min at 47° C. in thepresence of varying 16.6 concentrations. MDH refolding was initiated at30° C. by adding an ATP regenerating system (2 mM ATP; 3 mM PEP; 20ng/ml pyruvate kinase) and various chaperone combinations made up fromKJE (1 μM DnaK; 0.2 μM DnaJ; 0.1 μM GrpE) and ClpB (1.5 μM). The resultsfor these experiments are shown in FIG. 8.

Experiments were also carried out in which 1 μM MDH was denatured inbuffer A (50 mM Tris pH 7.5; 150 mM KCl; 20 mM MgCl₂) for 30 min at 47°C. in the absence or presence of 0.5 μM 16.6. MDH refolding wasinitiated at 30° C. by adding an ATP regenerating system (2 mM ATP; 3 mMPEP; 20 ng/ml pyruvate kinase) and the DnaK system (1 μM DnaK; 0.2 μMDnaJ; 0.1 μM GrpE) in the presence of varying ClpB concentrations asindicated. The results for these experiments are shown in FIG. 9.

Results:

All the sHSPs tested formed complexes with heat-denatured proteinsubstrates such as malate dehydrogenase (MDH), firefly luciferase andalpha-glucosidase which represented small protein aggregates. The datashown in FIG. 5 show that ClpB strongly stimulates the DnaK-dependentrefolding of the thermolabile reporter protein malate dehydrogenase(MDH) from various soluble sHSP/MDH complexes. This stimulatory effectwas verified by analysis of the refolding of the substrates fireflyluciferase, citrate synthase and α-glucosidase from complexes with sHSP16.6 (shown in FIG. 6 and FIG. 7). Notably, the refolding of substratesby ClpB/DnaK from sHSP/substrate complexes was in general much fasterthan refolding from aggregated proteins generated by identicaldenaturation conditions in the absence of sHSPs (FIG. 7). The GroESLchaperone system was not able to refold any of the substrates testedfrom sHSP/substrate complexes, even in the presence of ClpB. HoweverGroESL was observed to increase the rates of substrate refolding in thepresence of DnaK or ClpB/DnaK, especially in case of MDH (FIG. 5). Table2 provides a summary of the results from these experiments: TABLE 2Refolding of thermolabile proteins from protein aggregates or solublesHsp/protein complexes Chaperones Substrate KJE KJE/ESL KJE/ClpBKJE/ClpB/ESL aggr. MDH 0.1 0.2 10.3 25.1 sHsp/MDH 4.0 9.9 8.5 27.5 aggr.α-glucosidase 0 0 1.73 2.27 sHsp/α-glucosidase 0.44 0.53 2.69 3.63 aggr.citrate synthase 0 0 0.06 0.1 sHsp/citrate synthase 0.12 0.22 0.4 0.63aggr. luciferase 0.01 n.d. 0.14 n.d. sHsp/luciferase 0.17 n.d. 0.48 n.d.Refolding rate (nM/min)MDH, α-glucosidase, citrate synthase and luciferase were denatured inthe absence or presence of a 4-fold excess of 16.6. Substrate refoldingwas initiated by addition of an# ATP-regenerating system and the indicated chaperone combinations(experimental details as described above). Maximal rates of substraterefolding were derived from the linear # phase of the time curves ofrecovered enzymatic activity.

On the basis of these results, we propose that sHSP/substrate complexesrepresent small protein aggregates and refolding of substrates from suchcomplexes relies on a disaggregation reaction mediated by the DnaKsystem alone, or much more efficiently by ClpB with the DnaK system.After their active extraction from the complex, unfolded substrates aresubsequently refolded by a chaperone network formed by the DnaK andGroESL systems.

In vivo the levels of sHSPs are often not sufficient to prevent proteinaggregation and sHSPs are usually found associated with proteinaggregates. We investigated whether the presence of sHSPs in proteinaggregates can facilitate their resolubilization and consequentlyincrease substrate refolding. To answer this question the amount ofsHSPs utilised in each experiment was titrated during the denaturationof MDH and the resulting consequences on DnaK or DnaK/ClpB-mediated MDHrefolding were investigated. Substiochiometric concentrations of Hsp16.6compared to MDH resulted in the formation of insoluble, turbid sHSP/MDHcomplexes which were, however, much smaller than MDH aggregates formedby denaturation in the absence of Hsp16.6 (Table 3). TABLE 3Characterisation of 16.6/MDH complexes Size determination Dynamic 16.6/lightscattering Static MDH Lightscattering Solubility Calculatedlightscattering Ratio intensity (%) (%) radius (nm) Mass (Da) 0 100 <1045 +/− 15 n.d. 0.25 68 <10 33.7 +/− 12.5 1.8E+07-7.0E07  0.5 37 18 31.5+/− 9   1.8E+07-7.0E+07 1 0 57 24 +/− 6  5.6E+07-1.5E+07 2 0 84 19 +/−5  2.3E+06-4.0E+06 4 0 92 14 +/− 5  1.5E+06-3.1E+061 μM MDH was denatured in buffer A (50 mM Tris pH 7.5; 150 mM KCl; 20 mMMgCl₂) for 30 min at 47° C. in the presence of varying 16.6concentrations,# given as 16.6/MDH ratio. Turbidity (light scattering intensity) offormed MDH aggregates was set at 100%. Solubility of native, untreatedMDH after centrifugation # (13.000 rpm, 15 min, 4° C.) was set 100%.Size of the different sHSP/substrate complexes were determined either bydynamic or static lightscattering # (coupled to gelfiltation)measurements. Both techniques were utilised in case of poorly solublesHSP/MDH complexes leading to characterization of a subpopulation of thecomplexes only.

Increasing Hsp16.6 concentrations increased the solubility and decreasedturbidity and size of sHSP/MDH complexes (Table 3). EfficientDnaK-dependent MDH refolding required the presence of soluble sHSP/MDHcomplexes created in the presence of high Hsp16.6 concentrations (FIG.8). In contrast ClpB/DnaK mediated MDH refolding did not show up such asevere dependency, however MDH activity was recovered at earliertimepoints if insoluble sHSP/MDH complexes instead of MDH aggregateswere used as starting material. This effect became much more severe, ifthe disaggregation potential of the ClpB/DnaK system was reduced bylowering the ClpB concentration (FIG. 9). The stimulatory effectsdescribed above were again observed when substoichiometricconcentrations of sHSPs were present during substrate denaturation (byheat), resulting in the formation of insoluble sHSP/substrate complexes.Thus the presence of sHSPs in insoluble protein aggregates cansignificantly facilitate aggregate resolublization by ClpB/DnaK.

The above example illustrates that refolding of substrates after theirClpB/DnaK mediated extraction from sHSP/substrate complexes is in mostcases stimulated by the GroESL chaperone system, indicating thatreleased, unfolded substrates are refolded by a chaperone network. Weconclude that sHSP function is coupled to ClpB/DnaK dependent proteindisaggregation and serves to prepare protein aggregates for fasterresolubilization.

Example 11 Investigation of the Effect of Small Heat Shock Proteins onthe Yield of Soluble Recombinant Proteins in vivo

Materials & Methods:

E. coli wild type or ΔibpAB or ΔdnaK mutant cells were grown at 30° C.to logarithmic phase and shifted to 45° C. for 30 min, followed by arecovery phase at 30° C. for 60 min. Protein aggregates were isolated atthe indicated timepoints and analyzed by SDS-PAGE. The results for theseexperiments are shown in FIG. 10.

E. coli wild type or ΔibpAB or ΔclpB or ΔibpAB ΔclpB double mutantstrains were grown at 30° C. to logarithmic phase. Cells were eithershifted directly to 50° C. or were preincubated at 42C. for 15 min.Various dilutions of stressed cells were plated on LB plates. After 18 hcolony numbers were counted and survival rates were calculated inrelation to determined cell numbers before 50° C. shock. The results forthese experiments are shown in FIG. 11.

Various dilutions (10⁻³ to 10⁻⁶) of the cultures were spotted on LBplates supplemented with the indicated IPTG concentrations and incubatedat 30° C., 37° C. or 42° C. for 18 h. The results for these experimentsare shown in FIG. 12.

Various strains of E. coli were grown overnight at 30° C. in thepresence of 500 μM IPTG. Cultures were washed twice with LB andinoculated for further growth at 30° C. in the presence of variousIPTG-concentrations (0, 25, 50, 100 μM) to logarithmic phase and shiftedto 42° C. for 30 min. Protein aggregates were isolated at the indicatedtimepoints and analyzed by SDS-PAGE. The results for these experimentsare shown in FIG. 13.

In the experiments described above in examples 6-9 we expressed in E.coli strain BL21 (DE3) several target proteins including 2C18, E8R, Tep3and Kringle with or without co-expression of different combinations ofthe chaperones GroELS, ClpB, DnaK, DnaJ and GrpE. The chaperonecombination which for each case yielded the highest levels of solubletarget proteins was taken as “control” (overproduction of KJE/ELS/B for2C18, Tep3, no chaperone overproduction for E8R and Kringle). To showthe solubilization effects of overproduction of IbpA/IbpB together withother chaperones we generated BL21(DE3) strains which carry plasmidsexpressing IPTG-regulatable genes encoding these same target proteinsand in addition plasmids expressing IPTG regulatable genes encodingIbpA/IbpB (lanes marked IbpAB in FIG. 14), IbpA/IbpB and GroELS (lanesmarked IbpAB+GroELS in FIG. 14), IbpA/IbpB and GroELS and DnaK/DnaJ/GrpEand ClpB (lanes marked IbpAB+compl. in FIG. 14). After IPTG inductionthe bacteria were cultured overnight at 20° C. and directly collected(I), or the IPTG was removed and the pellet re-suspended in fresh mediumand cultured for two additional hours without (N) or with 200 μg/ml ofchloramphenicol (C). For each combination the amount of soluble protein(after affinity purification of the target proteins in the soluble cellfractions) was identified on Coomassie-stained SDS-gels. The results forthese experiments are shown in FIG. 14.

Results:

E. coli mutant cells missing the sHSPs IbpA/B do not exhibit atemperature-dependent growth phenotpye (42° C.). However, we observedthat the resolubilization of protein aggregates, created by severe heattreatment (45° C.), was delayed in comparison to wild type cells (FIG.10). Additionally the survival rate (thermotolerance) of ΔibpAB mutantsat lethal temperatures (50° C.) was slightly reduced compared to wildtype (FIG. 11). Thermotolerance is linked to the ability of cells torescue aggregated proteins and consequently the observed reducedthermotolerance of ΔibpAB mutants is likely caused by a less efficientresolubilization of protein aggregates.

DnaK has been shown to be the major player in preventing proteinaggregation in E. coli at high temperatures. We therefore investigatedwhether IbpA/B function could become more important in the presence ofreduced DnaK levels, rendering E. coli cells more sensitive to proteinaggregation. In vivo depletion of DnaK was achieved by replacing theσ32-dependent promotor of the dnaKJ operon by an IPTG-inducible one.Reduced DnaK levels caused synthetic lethality in ΔibpAB mutant cells atelevated temperatures (37-42° C.). The same experiments performed in aΔclpB mutant strain and a ΔibpAB ΔclpB double knockout revealed anincreasing necessity for higher DnaK levels at elevated temperatures(FIG. 12). Especially in case of the ΔibpAB ΔclpB double knockout mutantstrain this phenotype was linked to severe protein aggregation upon heatshock to 42° C. (FIG. 13). Thus in vivo IbpA/B is necessary forefficient protein disaggregation, especially under conditions whichfavour protein aggregation and lower the disaggregation potential ofcells.

As shown in FIG. 14 and Table 4, the combined overproduction of IbpABwith ClpB, the DnaK system and the GroEL system, and with combinationsof these chaperones, increases the yield of soluble recombinant proteinproduced in E. coli cells. TABLE 4 Protein MW Organism Features IpbAB IFSerprotAg1 A. gambiae domain/fus ! Kringle 30 kD H. sapiens domain/fus !2C18 50 kD H. sapiens Fl/fus 2.5 22j21 72 kD H. sapiens Fl/fus 0 Tep3 70kD A. gambiae domain/fus/secr 3.5 Tep4 68 kD A. gambiae domain/fus/secr0 XklpA3 73 kD X. laevis domain/fus/cyt 0 E8R1 58 kD Vaccinia virusFl/membr/fus 3.5 BtKe 55 kD H. sapiens domain/cyt 0Nine proteins were tested for the effects of IpbAB co-expression on thelevel of soluble target proteins produced in E. coli cells. Theincrement factor (IF) defines the fold increase (in the best condition,being either I, N or C;# see above for definition) in amount of soluble protein due to IpbABco-expression with respect to the controls (the best conditionsidentified from examples 6-9). # ! denotes that the IpbAB-dependentexpression of soluble proteins occurred which could not be produced insoluble form before. Thus, in 5 of the nine cases tested, theoverproduction of IbpA/IbpB further increasesd the yield of targetproteins.

Thus, these in vivo data are consistent with the results obtained invitro. Firstly, the yields of soluble recombinant protein produced in E.coli cells can be increased in several cases tested when IbpA/IbpB isoverproduced alone or together with various combinations of the DnaK andGroELS systems and ClpB. Secondly, E. coli ΔibpAB mutant cells missingIbpA/B exhibited a delayed protein disaggregation after heat shock (45°C.) and a reduced survival rate at lethal temperatures (50° C.) comparedto wild type cells. IbpA/B function became essential at elevatedtemperatures (37-42° C.) in the presence of reduced DnaK levels,conditions which favour protein aggregation and reduce thedisaggregation potential of the cells.

In summary, the above Examples 10 and 11 show that small heat shockproteins (sHSPs) co-operate with other chaperones, in particular withthe ClpB chaperone, the DnaK chaperone system and the GroEL chaperonesystem, to solubilize and refold aggregation-prone proteins. Thisproperty can be exploited to increase the yield of soluble recombinantproteins produced in E. coli and other cells, and can be used for the invitro production of soluble recombinant protein. In particular, thecombined overproduction of IbpAB with ClpB, the DnaK system and theGroEL system, and with combinations of these chaperones, increases theyield of soluble recombinant protein produced in E. coli cells. However,the teaching provided by these experiments is of much broader importancesince all the proteins involved in this folding reaction are members oflarge protein families with members among prokaryotes and eukaryotes(IbpA and IbpB are members of the family of sHSPs which includesalpha-cristallins; ClpB is member of the AAA protein family whichinclude Hsp104; DnaK is member of the Hsp70 family; DnaJ is member ofthe DnaJ (Hsp4O) family; GrpE is member of the GrpE family; GroEL ismember of the Hsp60 family; GroES is member of the GroES family). It isexpected that the other members of the involved protein families cansubstitute for the E. coli members in protein folding reactions. Infact, we present biochemical data that the sHSP of Synechocystis,Hsp16.6, can increase the efficiency of protein refolding in cooperationwith the E. coli chaperones ClpB, DnaK, DnaJ, GrpE, and GroELS.Furthermore, since ClpB is a homolog of the S. cerevisiae Hsp104, achaperone implicated in the generation and prevention of formation ofamyloid fiber formation, it is also possible that our finding that thesHSPs co-operate with ClpB and the DnaK and GroEL systems in proteinfolding has implications on the formation or treatment of amyloid fibersin eukaryotic cells, and diseases in which such fibers are implicated.

Finally, it was found that the IbpA/B, ClpB and the DnaK systems actcooperatively to reverse protein aggregation.

To elucidate the functional interplay between IbpA/B, KJE and ClpB inthe protein quality control network more precisely, we determined thedegree of protein aggregation in ΔibpAB, ΔclpB and ΔibpAB ΔclpB mutantsthat have KJ adjusted to various levels. Since ClpB and IbpA/B do notprevent protein aggregation in vivo (Mogk et al., 1999), increasedamounts of aggregated proteins in the respective mutants would indicatea less efficient protein disaggregation. At 30° C. no proteinaggregation was detectable for all tested mutant strains, even in cellswith greatly reduced KJ levels. After a 30 min incubation at 42° C., 5%of cellular proteins aggregated in all mutant cells, provided that IPTGwas omitted from the growth medium. Increasing KJ levels (by addition ofIPTG) reduced the amount of aggregated proteins in each strain, but todifferent degrees dependent on the mutant background. While 50 μM IPTGin the growth medium was sufficient to eliminate aggregates in ΔibpABcells, 2% and 5% of total proteins still aggregated in ΔclpB and ΔibpABΔclpB mutant cells, respectively. Even in presence of DnaK/DnaJ levelscorresponding to heat shock conditions (100 μM IPTG), 2% of cellularproteins remained aggregated in ΔibpAB ΔclpB mutant cells.

These findings are in complete agreement with the hierarchialcomplementation of growth defects of these mutant cells at hightemperatures and demonstrate the cooperative action of IbpA/B and ClpBin the KJE-mediated removal of protein aggregates in vivo.

Example 12 Bacteria Co-Transformed with Recombinant Proteins andChaperones Cloned in Independent Plasmids are Suitable for ExpressionTuning

This example describes a system based on three vectors, where two areunder IPTG regulation and enable the recombinant expression of sixchaperones, and the third one is arabinose-inducible and harbours thesequence for the recombinant target protein of interest. In such a way,the independent induction and the level of expression of both chaperonesand target protein was possible. The data showed that the expressionleakage from pET vectors was prevented by the introduction of furtherplasmids in the cell and that the recombinant proteins compete for theirexpression. In fact, the high rate induction of one of them could switchoff the accumulation of the other recombinant proteins. The firstinformation was used to maximise the expression of toxic proteins whilethe cross-inhibition among recombinant proteins was exploited tomodulate and optimise the target protein expression and to induce thechaperone-assisted in vivo re-folding of aggregated target protein.

Cloning and Transformation Procedures.

Chaperone proteins were expressed as described above. For expression oftarget protein, the sequences corresponding to GTR1 (O 00582) and themotor regions of Xklp3A and Xklp3B (AJ 311602; CAA 08879) were cloned inpTrcHis vector (trc promoter and ColE1 replication origin), Tep3(unpublished sequence from A. gambiae) was cloned in pGEX (tac promoterand pBR322 replication origin) and E8R (NP 063710) in pGAT (lac promoterand pUC replication origin). The sequences for GTR1, Tep3, E8R, theXklp3A and B C-terminal regions of the coil-coiled domains and a domainof Btk (O 06187) were cloned in pBAD. pET24d and pETM60. The Xklp3A andB C-terminal regions of the coil-coiled domains were also cloned in thepolycistronic vector pST39 (Tan, 2001).

Cell Cultures.

Single colonies from the transformed cells were used to inject 3 mL ofLB medium. Liquid cultures were incubated initially at 37° C.,successively transferred to 30° C. or 20° C., induced at an OD₆₀₀ of 0.8and grown 3 hours or overnight, respectively. Variations of timing andconcentration combinations used in the experiments with bacteria hostingboth IPTG and arabinose regulated expression vectors are described caseby case in the results.

Protein Purification and Yield Evaluation.

Frozen bacterial pellets corresponding to 0.5 mL of culture werere-suspended in 350 μL of 20 mM Tris HCl, pH 8.0, 2mM PMSF, 0.05% TritonX-100 and 1 mg/mL lisozyme and incubated on ice for 30 min, withperiodic stirring. The suspension was sonicated in water for 5 minutes,pelleted in a minifuge, the supernatant was added to 20 μL of pre-washedNi-NTA magnetic agarose beads (Qiagen) and incubated further 30 minunder agitation before being removed. Beads were washed 30 min with 20mM K-phosphate buffer, pH 7.8, 300 mM NaCl, 20 mM imidazole, 8%glycerol, 0.2% Triton X-100 and later with PBS buffer plus 0.05% TritonX-100. Finally they were boiled in 12 μL SDS sample buffer and thesamples loaded onto a SDS PAGE using a Pharmacia rninigel system.Proteins were detected after coloration with Simply Blue Safestain(Invitrogen) following the manufacturer's instructions and the gels wererecorded using a Umax Astra 4000U scanner. Proteins were quantifiedanalysing the gel bands with the public domain NIH Image program(developed at the U.S. National Institutes of Health and available onthe Internet at http://rsb.info.nih.gov/nih-image/).

Results and Discussion

Initially we transformed bacteria with chaperone-carrying plasmids andin a second step these cells were re-transformed with a plasmidharbouring the target protein. The expression of all the plasmids wasunder IPTG regulation. The cell co-transformation with three plasmidsselected using different antibiotic resistances induced a 20% decreaseof the cell growth rate; however, succeeded even in the case in whichtwo plasmids (PGAT and pBAD) shared the same replication origin pUC.

Bacteria transformed with two low copy number plasmids derived from pDM1and harbouring different chaperone genes expressed the correspondingproteins at very high level (FIG. 15A). Nevertheless, the intensity ofthe bands separated in SDS-gel indicated 10 that the expression of thetarget protein GTR1 cloned into the pTrcHis vector strongly inhibitedthe chaperone accumulation (compare FIGS. 15A and 15B) so that ClpB wasno more detectable in the bacterial homogenate (FIG. 15B). In contrast,the expression of the target protein Btk by the leaking vector pET24d inthe absence of the inducer IPTG was strongly repressed when achaperone-containing plasmid was co-transformed in the host cell (FIG.15C). These results suggest that there are two independent kinds ofinteraction raising from the presence of different plasmids in the samecell. The first one involves the plasmids and is independent from theirprotein products. In fact, the IPTG-independent expression of Btk clonedin a pET24d expression vector was prevented also in the case of theco-transformation with an empty pDM1 vector (FIG. 15D). A recent paperreports that the introduction of heterologous vectors has been shown toinduce stress responses and inhibit biomass production in S. cerevisiaeeven though they were empty or non-induced (Görgens et al., 2001,Biotechnol. Bioeng. 73, 238-245).

The expression-leakage control obtained by co-transformation with moreplasmids at once can be useful in the case of the expression of toxicproteins or when the leakage rate is so high to impair the normal cellfunction. At least one experience in the frame of this work indirectlysupports this hypothesis. A polycistronic plasmid (Tan, 2001) has beenused for expressing a complex between the C-terminal end of thecoil-coiled regions of Xklp3 chain A and chain B. No colony grew usingBL21 (DE3) bacteria when we tried to transform them with thepolycistronic plasmid. Cells co-transformed with chaperone plasmids wereefficiently transformed with the polycistronic vector and gave colonies.Colonies grew also when the polycistronic vector was transformed intopLysS strain cells and 1% glucose was added to the growth medium totightly control any expression leakage. However, the bacterial yield was60% less (data not shown) and the purified protein decreased of morethan 80% (FIG. 16).

Beside the case of plasmid interaction our results seem to indicate thatthe cell machinery involved in the protein production was challenged bythe contemporary over-expression of too many recombinant proteins. Theresults of FIG. 15A and 15B could be interpreted either as anoverwhelming accumulation of target protein transcripts that inhibitsthe chaperone expression rate or a competition for the RNA polymerase.Such a competition has been described in E. coli between metabolic andrecombinant genes (Schweder et al., 2002, Appl. Microbiol. Biotechnol.58, 330-337) while recombinant and cell mRNAs could compete attranscriptional level in yeast (Görgens et al., 2001, Biotechnol.Bioeng. 73, 238-245). We observed that in case of co-transformation theeffect of competition seems proportional to the estimated copy number ofthe target protein plasmid and independent on the promoter used (datanot shown). In fact, recombinant vectors hosting the target protein withboth T7 and lac promoters could inhibit the chaperone expression.Therefore, a competition at the transcriptional level would be ruled outin our system. The existence of a limit of total protein expression canhave important consequences in the case in which recombinant chaperonesare co-transformed to boast the production of a target protein. In fact,a too high level of expression of the latter could automatically inhibitthe chaperone expression levels and, therefore, limit or prevent theirpositive folding effect.

An alternative method has been envisaged in which chaperones and targetproteins were cloned in vectors in which their expression was underdifferent regulation systems. This enables the independent induction ofchaperone and target protein expression and would allow exploitation ofthe chaperone-dependent folding improvement of the target proteinavoiding any shortcomings due to contemporary co-expression. A logicalapproach seemed to induce the accumulation of the chaperones and thentrigger the target protein expression in a cell with boasted foldingmachinery.

In a first set of experiments the GroELS chaperones were expressed bymeans of an arabinose-regulated vector (Castanié et al., 1997, Anal.Biochem. 254, 150-152) and the IPTG-dependent target proteins wereinduced after 30 minutes. The results did not show a significantincrease of soluble target proteins and no improvement was detectedvarying incubation times and inducer concentrations (data not shown).

In a second attempt the target proteins were cloned intoarabinose-regulated vectors while five different IPTG-dependentchaperone combinations were compared. Such an expression system mostlyresulted in an increased yield of the soluble target proteins (see Table5). TABLE 5 Chaperone-dependent yield improvement of soluble targetproteins. Clones corresponding to the target proteins wereco-transformed with the different chaperone combinations and culturedaccording to the best among the conditions reported in FIG. 17. Theimprovement factor enabled by chaperone co-expression indicates theratio between the highest yield of soluble target protein obtained usingcells co-transformed with chaperones and its amount recovered from cellsnot hosting recombinant chaperones; the symbol ∞ means that no solubletarget protein was expressed in absence of chaperones. The targetproteins were expressed in arabinose-regulated pBAD vectors and thedifferent chaperone combinations listed in material and methods wereinduced by IPTG addition. Protein MW Organism Improvement Factor GTR1 40kD S. cerevisiae 3 Btkp 55 kD H. sapiens 3 Xklp3A 62 kD X. laevis ∞Xklp3B 40 kD X. laevis 9 Tep3 70 kD A. gambiae 4 E8R 32 kD Vacciniavirus 0

The optimal chaperone combination (FIG. 17A) and the expressionconditions were specific for each target protein. The complexity of theinteractions among the different recombinant proteins is illustrated inthe experiments summarised in FIG. 17B and 17C. Soluble GTR1accumulation was induced at a similar level by both 0.5 and 1.5 mg/mL ofarabinose (FIG. 17B, lanes 1 and 2). The co-expression of low amounts ofK+J+E+ClpB+GroELS chaperones induced by 0.02 mM IPTG stimulated theaccumulation of soluble GTR1 whose expression was induced by 0.5 mg/mLof arabinose (FIG. 17B, lane 4).

Nevertheless, the amount of the soluble target protein decreased ifIPTG-dependent chaperones were allowed to accumulate before thearabinose-dependent induction of GTR1 (FIG. 17B, compare lanes 4 and 5).The same pattern of inhibition of soluble GTR1 was observed when higherchaperone expression was induced by ten-fold higher IPTG concentrationbut, in such a case, the absolute amount of soluble GTR1 was stronglyreduced (lanes 6 and 7). These data confirm the existence of acompetition among the products of different recombinant plasmids. Inthis case, both plasmids use the cell RNA polymerase and, therefore, itis not possible to distinguish between competition at the transcriptionor translation level. Nevertheless, the accumulation of soluble GTR1induced at low level of arabinose is progressively inhibited by anincreasing amount of available chaperones (FIG. 17B). The inhibitorychaperone accumulation was obtained with both higher IPTG concentrationand longer time of induction before the arabinose-dependent induction ofGTR1. When we repeated the same experiments using 1.5 mg/mL of arabinoseto induce a higher GTR1 expression the results were reversed (FIG. 17B,compare lanes 8-11 and 4-7). As a matter of fact the higher arabinoseconcentration enabled a strong accumulation of GTR1; however, theincreasing amounts of expressed chaperones did not reach a levelcritical for competition but could provide a more stabilisingenvironment for GTR1.

The conclusions from this work are that chaperones can positivelycontribute to GTR1 accumulation. Nevertheless, a ratio among thetranscripts seems to be important for avoiding detrimental competitionat the translation level. The parameters involved are the rate ofinduction of both chaperone and target genes and the time in whichchaperones can accumulate before the target protein is induced.

Recently, it has been showed that recombinant proteins precipitated inaggregates could be re-solubilised in vivo. Aggregate re-folding wasinduced after that translation inhibition made available foldases andchaperones otherwise employed in metabolic folding (Carrió andVillaverde, 2001, FEBS Letts. 489, 29-33). We applied this idea to oursystem in which the accumulation of recombinant chaperones was possible.

The expression of coiled-coil Xklp3B was induced overnight at 0.5 mg/mLof arabinose (FIG. 17C, lane 1). The amount of recovered soluble proteinwas low and inhibited or almost completely prevented when chaperones(K+J+E+GroELS+ClpB) expression was IPTG-induced together or beforearabinose addition (FIG. 17C, lanes 2 and 3). These data confirm theresults collected using GTR1 and explained considering a competitionamong the recombinant proteins (FIG. 17B). In contrast, the removal ofthe arabinose-containing medium and the addition of fresh medium pluschloramphenicol had a positive effect on the amount of recovered solubleprotein (FIG. 17C, lane 4). Apparently, the standard cellular foldingmachinery is, therefore, sufficient to partially re-fold the aggregatedrecombinant target protein. Nevertheless, a strong re-solubilisationimprovement of the target protein was observed only when arabinose wasremoved, the pellet was re-suspended in fresh medium andchaperone-expression was induced by 0.2 mM IPTG addition (FIG. 17C, lane5). A similar improvement at a slightly lower extent was obtained by thesimple addition of a sufficiently high amount of IPTG (0.2 mM) to thearabinose-containing medium (FIG. 17C, lane 6). Therefore, it seems thatit is possible to exploit the inhibitory effect of an overwhelmingchaperone expression on the arabinose-regulated target protein to switchthe system from Xklp3B to chaperone expression. Then, in conditions thatinhibit the further expression of Xklp3B (comments to FIG. 17B), theavailable chaperones induce the re-folding of the already aggregatedtarget protein without the need to remove the arabinose from the medium.

The collected results provide new information concerning theco-transformation of more than one recombinant proteins and confirm thatchaperone co-transformation can increase the amount of soluble targetprotein. They also indicate that interactions among transformed plasmidsand among corresponding proteins need to find an equilibrium in the hostcell to optimise the co-transformation benefit. In fact, it seems thatchaperones can somehow compete with the target protein, meaning thatsome care is required to optimise each candidate system, although thisis well within the ambit of the skilled worker. Nevertheless, thereciprocal expression inhibition between target protein and chaperonescan be exploited to tune the expression rate and improve the amount ofsoluble target protein. We must only be aware that the conditions needto be optimised since the accumulation rate is specific for eachrecombinant protein.

1. A method for the expression of a recombinant protein of interest, said method comprising: a) culturing a host cell which expresses: i) one or more genes encoding the recombinant protein(s) of interest; ii) at least two genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs under conditions suitable for protein expression; and b) separating said recombinant protein of interest from the host cell culture.
 2. A method according to claim 1, wherein the genes selected in step a) ii) include DnaK, DnaJ and GrpE or homologs thereof.
 3. A method according to claim 2, wherein the genes selected in step a) ii) additionally include ClpB or a homolog thereof.
 4. A method according to claim 1, wherein the genes selected in step a) ii) include GroES and GroEL or homologs thereof.
 5. A method according to claim 4, wherein the genes selected in step a) ii) include the DnaK, DnaJ, GrpE, ClpB, GroES and GroEL genes or homologs thereof.
 6. A method for the expression of a recombinant protein of interest, said method comprising: a) culturing under conditions suitable for protein expression a host cell which expresses: i) one or more genes encoding one or more recombinant protein(s) of interest; ii) one or more genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs; iii) one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family small heatshock proteins of the IbpB family and homologs thereof; and (b) separating said recombinant protein of interest from the host cell culture.
 7. A method according to claim 1 wherein the levels of the respective chaperone proteins are controlled.
 8. A method according to claim 7, wherein said levels of chaperone proteins are controlled by expressing the genes encoding the respective chaperone proteins from different promoters.
 9. A method according to claim 7, wherein the respective chaperone proteins are expressed using expression systems of different strength.
 10. A method according to claim 7, wherein said chaperone proteins are over-expressed relative to the expression levels that occur naturally in non-recombinant cells.
 11. A method according to claim 1, wherein the levels of the chaperone proteins relative to the recombinant protein(s) of interest are controlled by expressing the genes encoding the respective proteins from different promoters or by using different polymerases.
 12. A method according to claim 1, wherein in culturing step a) of the method, a block in protein synthesis is imposed, for example, by the addition of an effective amount of a protein synthesis inhibitor to the culture system, once a desired level of recombinant protein of interest has accumulated.
 13. A method according to claim 12, wherein the chosen protein synthesis inhibitor is chloramphenicol, tetracycline, gentamycin or streptomycin.
 14. A method according to claim 1, wherein in culturing step a) of the method, a reduction in gene transcription is imposed, for example, by removal of any agents that are effective to induce recombinant protein expression, or via the addition of a transcription blocking compound, once a desired level of recombinant protein of interest has accumulated
 15. A method for the expression of a recombinant protein of interest, said method comprising: a) culturing a host cell which expresses: i) one or more genes encoding the recombinant protein(s) of interest; ii) one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs; under conditions suitable for protein expression; b) imposing a block in protein synthesis, for example, by the addition of an effective amount of a protein synthesis inhibitor to the culture system, once a desired level of recombinant protein of interest has accumulated; and c) separating said recombinant protein of interest from the host cell culture.
 16. A method for the expression of a recombinant protein of interest, said method comprising: a) culturing a host cell which expresses: i) one or more genes encoding the recombinant protein(s) of interest; ii) one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs; under conditions suitable for protein expression; b) imposing a reduction in gene transcription, for example, by removal of any agents that are effective to induce recombinant protein expression, or via the addition of a transcription blocking compound, once a desired level of recombinant protein of interest has accumulated; and c) separating said recombinant protein of interest from the host cell culture.
 17. A method according to claim 15, wherein said host cells additionally expresses one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family and/or the IbpB family and/or their homologs.
 18. A method according claim 14, wherein in step a) ii), a combination of chaperone proteins is expressed.
 19. A method according to claim 15, wherein the chosen protein synthesis inhibitor is chloramphenicol, tetracycline, gentamycin or streptomycin.
 20. A method according to claim 1, wherein said cultured host cell is a prokaryotic cell, such as an E. coli cell, a Lactococcus cell, a Lactobacillus cell or a Bacillus subtilis cell, or a eukaryotic cell such as a yeast cell, for example a Pichia or Saccharomyces yeast cell, or an insect cell, for example after baculoviral infection.
 21. A method according to claim 1, wherein an optimised yield of said recombinant protein of interest is manifested by increasing the level of de novo protein folding.
 22. A method according to claim 1, wherein an optimised yield of said recombinant protein of interest is manifested by increasing the level of in vivo refolding of aggregated, or misfolded soluble, recombinant protein.
 23. A method according to claim 1, wherein an optimised yield of said recombinant protein of interest is manifested by increasing the level of in vitro refolding of aggregated, or misfolded soluble, recombinant protein.
 24. A method according to claim 20, wherein an optimised yield of said recombinant protein is manifested by increasing the level of de novo protein folding in combination with an increased level of in vivo protein refolding and/or in vitro protein refolding.
 25. A method according to claim 21, wherein said increased level of folding or re-folding results in increased solubility of the recombinant protein of interest.
 26. A method according to claim 21, wherein said increased level of folding or re-folding results in increased activity of the recombinant protein of interest.
 27. A method for increasing the degree of refolding of a recombinant protein of interest, said method comprising adding a composition containing a chaperone protein to a preparation of the recombinant protein of interest in vitro.
 28. A method according to claim 27, wherein a combination of chaperone proteins is added to the preparation of the recombinant protein of interest.
 29. A method according to claim 27, wherein the preparation of the recombinant protein of interest is a preparation of soluble recombinant protein that has been precipitated in vivo.
 30. A method according to claim 27, wherein the preparation of the soluble recombinant protein of interest is a preparation of in vitro precipitated recombinant protein.
 31. A method according claim 27, wherein said composition containing the chaperone protein(s) is added after removal of any agents that are effective to induce soluble recombinant protein expression or after addition of a transcription blocking compound.
 32. A method according to claim 27, additionally comprising the step of imposing a block in protein synthesis, such as by the addition of an effective amount of a protein synthesis inhibitor to the culture system.
 33. A method according to claim 32, wherein the chosen protein synthesis inhibitor is chloramphenicol, tetracycline, gentamycin or streptomycin.
 34. A method according claim 1, wherein the refolding temperature and time course of refolding are controlled.
 35. A method according to claim 27, additionally comprising the use of one or more proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the heatshock proteins of the IbpB family and homologs thereof.
 36. The use of one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof, in the manufacture of a medicament for the treatment of disease in which the presence of aggregated proteins are implicated.
 37. The use of one or more selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof, in the manufacture of a medicament for the treatment of disease in which the presence of aggregated proteins are implicated.
 38. A method of treating a patient suffering from a disease in which the presence of aggregated proteins is implicated, comprising administering one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof.
 39. A method of treating a patient suffering from a disease in which the presence of aggregated proteins is implicated, comprising administering one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof.
 40. The method of claim 38, wherein the disease is late or early onset Alzheimer's disease, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, or a spongiform encephalopathy.
 41. A method according to claim 2, wherein the genes selected in step a) ii) include GroES and GroEL or homologs thereof
 42. A method according to claim 3, wherein the genes selected in step a) ii) include GroES and GroEL or homologs thereof.
 43. A method according to claim 41, wherein the genes selected in step a) ii) include the DnaK, DnaJ, GrpE, ClpB, GroES and GroEL genes or homologs thereof.
 44. A method according to claim 42, wherein the genes selected in step a) ii) include the DnaK, DnaJ, GrpE, ClpB, GroES and GroEL genes or homologs thereof.
 45. A method according to claim 16, wherein said host cells additionally expresses one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof.
 46. The method of claim 39, wherein the disease is late or early onset Alzheimer's disease, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, or a spongiform encephalopathy. 